Chemical sensor array having multiple sensors per well

ABSTRACT

In one embodiment, a device is described. The device includes a material defining a reaction region. The device also includes a plurality of chemically-sensitive field effect transistors have a common floating gate in communication with the reaction region. The device also includes a circuit to obtain respective output signals from the chemically-sensitive field effect transistors indicating an analyte within the reaction region.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 14/293,247filed Jun. 2, 2014; which claims the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application No. 61/833,375 filed Jun. 10, 2013. Theentire contents of the aforementioned applications are incorporated byreference herein.

BACKGROUND

The present disclosure relates to sensors for chemical analysis, and tomethods for manufacturing such sensors.

A variety of types of chemical sensors have been used in the detectionof chemical processes. One type is a chemically-sensitive field effecttransistor (chemFET). A chemFET includes a source and a drain separatedby a channel region, and a chemically sensitive area coupled to thechannel region. The operation of the chemFET is based on the modulationof channel conductance, caused by changes in charge at the sensitivearea due to a chemical reaction occurring nearby. The modulation of thechannel conductance changes the threshold voltage of the chemFET, whichcan be measured to detect and/or determine characteristics of thechemical reaction. The threshold voltage may for example be measured byapplying appropriate bias voltages to the source and drain, andmeasuring a resulting current flowing through the chemFET. As anotherexample, the threshold voltage may be measured by driving a knowncurrent through the chemFET, and measuring a resulting voltage at thesource or drain.

An ion-sensitive field effect transistor (ISFET) is a type of chemFETthat includes an ion-sensitive layer at the sensitive area. The presenceof ions in an analyte solution alters the surface potential at theinterface between the ion-sensitive layer and the analyte solution, dueto the protonation or deprotonation of surface charge groups caused bythe ions present in the analyte solution. The change in surfacepotential at the sensitive area of the ISFET affects the thresholdvoltage of the device, which can be measured to indicate the presenceand/or concentration of ions within the solution.

Arrays of ISFETs may be used for monitoring chemical reactions, such asDNA sequencing reactions, based on the detection of ions present,generated, or used during the reactions. See, for example, U.S. Pat. No.7,948,015 to Rothberg et al., which is incorporated by reference herein.More generally, large arrays of chemFETs or other types of chemicalsensors may be employed to detect and measure static and/or dynamicamounts or concentrations of a variety of analytes (e.g. hydrogen ions,other ions, compounds, etc.) in a variety of processes. The processesmay for example be biological or chemical reactions, cell or tissuecultures or monitoring neural activity, nucleic acid sequencing, etc.

An issue that arises in the operation of large scale chemical sensorarrays is the susceptibility of the sensor output signals to noise.Specifically, the noise affects the accuracy of the downstream signalprocessing used to determine the characteristics of the chemical and/orbiological process being detected by the sensors.

It is therefore desirable to provide devices including low noisechemical sensors, and methods for manufacturing such devices.

SUMMARY

In one embodiment, a device is described. The device includes a materialdefining a reaction region. The device also includes a plurality ofchemically-sensitive field effect transistors have a common floatinggate in communication with the reaction region. The device also includesa circuit to obtain individual output signals from thechemically-sensitive field effect transistors indicating an analytewithin the reaction region.

In another embodiment, a method for manufacturing a device is described.The method includes forming a material defining a reaction region. Themethod further includes forming a plurality of chemically-sensitivefield effect transistors having a common floating gate in communicationwith the reaction region. The method further includes forming a circuitto obtain individual output signals from the chemically-sensitive fieldeffect transistors indicating an analyte within the reaction region.

Particular aspects of one more implementations of the subject matterdescribed in this specification are set forth in the drawings and thedescription below. Other features, aspects, and advantages of thesubject matter will become apparent from the description, the drawings,and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a block diagram of components of a system for nucleicacid sequencing.

FIG. 2 illustrates cross-sectional and expanded views of a portion of anintegrated circuit device and flow cell.

FIG. 3 illustrates a schematic diagram of a portion of the integratedcircuit device 100 including a sensor array having multiple chemicalsensors coupled to the same reaction region.

FIG. 4 is a flow chart of an example process for calculating a resultantoutput signal for a group of chemical sensors coupled to a singlereaction region.

FIG. 5 illustrates a cross-sectional view of portions of two groups ofchemical sensors and their corresponding reaction regions according to afirst embodiment.

FIGS. 6 to 10 illustrate stages in a manufacturing process for forming adevice including multiple chemical sensors coupled to the same reactionregion according to a first embodiment.

FIG. 11 illustrates a cross-sectional view of portions of two groups ofchemical sensors and their corresponding reaction regions according to asecond embodiment.

FIGS. 12 to 14 illustrate stages in a manufacturing process for forminga device including multiple chemical sensors coupled to the samereaction region according to a second embodiment.

FIGS. 15 to 18 illustrate stages in a manufacturing process for forminga device including multiple chemical sensors coupled to the samereaction region according to a third embodiment.

FIGS. 19 to 21 illustrate stages in a manufacturing process for forminga device including multiple chemical sensors coupled to the samereaction region according to a fourth embodiment.

DETAILED DESCRIPTION

A chemical detection device is described that includes multiple chemicalsensors for concurrently detecting a chemical reaction within the same,operationally associated reaction region. The multiple sensors canprovide redundancy, as well as improved accuracy in detectingcharacteristics of the chemical reaction.

By utilizing multiple chemical sensors to separately detect the samechemical reaction, the individual output signals can be combined orotherwise processed to produce a resultant, low noise output signal. Forexample, the individual output signals can be averaged, such that thesignal-to-noise ratio (SNR) of the resultant output signal is increasedby as much as the square root of the number of individual outputsignals. In addition, the resultant output signal can compensate fordifferences among the values of the individual output signals, caused byvariations in chemical sensor performance which could otherwisecomplicate the downstream signal processing. As a result of thetechniques described herein, low-noise chemical sensor output signalscan be provided, such that the characteristics of reactions can beaccurately detected.

FIG. 1 illustrates a block diagram of components of a system for nucleicacid sequencing according to an exemplary embodiment. The componentsinclude a flow cell 101 on an integrated circuit device 100, a referenceelectrode 108, a plurality of reagents 114 for sequencing, a valve block116, a wash solution 110, a valve 112, a fluidics controller 118, lines120/122/126, passages 104/109/111, a waste container 106, an arraycontroller 124, and a user interface 128. The integrated circuit device100 includes a microwell array 107 of reaction regions overlying groupsof chemical sensors of a sensor array as described herein. The flow cell101 includes an inlet 102, an outlet 103, and a flow chamber 105defining a flow path of reagents over the microwell array 107.

The reference electrode 108 may be of any suitable type or shape,including a concentric cylinder with a fluid passage or a wire insertedinto a lumen of passage 111. The reagents 114 may be driven through thefluid pathways, valves, and flow cell 101 by pumps, gas pressure, orother suitable methods, and may be discarded into the waste container106 after exiting the outlet 103 of the flow cell 101. The fluidicscontroller 118 may control driving forces for the reagents 114 and theoperation of valve 112 and valve block 116 with suitable software.

The microwell array 107 includes reaction regions, also referred toherein as microwells, which are operationally associated with chemicalsensors of the sensor array. As described in more detail below, eachreaction region is operationally associated with multiple chemicalsensors suitable for detecting an analyte or reaction of interest withinthat reaction region. These multiple chemical sensors can provideredundancy, as well as improved detection accuracy. The microwell array107 may be integrated in the integrated circuit device 100, so that themicrowell array 107 and the sensor array are part of a single device orchip.

In exemplary embodiments described below, groups of four chemicalsensors are coupled to each of the reaction regions. Alternatively, thenumber of chemical sensors operationally associated with a singlereaction region may be different than four. More generally, two or morechemical sensors may be operationally associated with a single reactionregion.

The flow cell 101 may have a variety of configurations for controllingthe path and flow rate of reagents 114 over the microwell array 107. Thearray controller 124 provides bias voltages and timing and controlsignals to the integrated circuit device 100 for reading the chemicalsensors of the sensor array as described herein. The array controller124 also provides a reference bias voltage to the reference electrode108 to bias the reagents 114 flowing over the microwell array 107.

During an experiment, the array controller 124 collects and processesindividual output signals from the chemical sensors of the sensor arraythrough output ports on the integrated circuit device 100 via bus 127.As described in more detail below, this processing can includecalculating a resultant output signal for a group of sensors as afunction of the individual output signals from the chemical sensors inthe group. The array controller 124 may be a computer or other computingmeans. The array controller 124 may include memory for storage of dataand software applications, a processor for accessing data and executingapplications, and components that facilitate communication with thevarious components of the system in FIG. 1.

In the illustrated embodiment, the array controller 124 is external tothe integrated circuit device 100. In some alternative embodiments, someor all of the functions performed by the array controller 124 arecarried out by a controller or other data processor on the integratedcircuit device 100. In yet other embodiments, a combination of resourcesinternal and external to the integrated circuit device 100 is used toobtain the individual output signals and calculate the resultant outputsignal for a group of sensors using the techniques described herein.

The value of a resultant output signal for a group of chemical sensorsindicates physical and/or chemical characteristics of one or morereactions taking place in the corresponding reaction region. Forexample, in an exemplary embodiment, the values of the resultant outputsignals may be further processed using the techniques disclosed inRearick et al., U.S. patent application Ser. No. 13/339,846, filed Dec.29, 2011, based on U.S. Prov. Pat. Appl. Nos. 61/428,743, filed Dec. 30,2010, and 61/429,328, filed Jan. 3, 2011, and in Hubbell, U.S. patentapplication Ser. No. 13/339,753, filed Dec. 29, 2011, based on U.S.Prov. Pat. Appl. No. 61/428,097, filed Dec. 29, 2010, each of which areincorporated by reference herein.

The user interface 128 may display information about the flow cell 101and the output signals received from chemical sensors of the sensorarray on the integrated circuit device 100. The user interface 128 mayalso display instrument settings and controls, and allow a user to enteror set instrument settings and controls.

The fluidics controller 118 may control delivery of the individualreagents 114 to the flow cell 101 and integrated circuit device 100 in apredetermined sequence, for predetermined durations, at predeterminedflow rates. The array controller 124 can then collect and analyze theoutput signals of the chemical sensors indicating chemical reactionsoccurring in response to the delivery of the reagents 114.

During the experiment, the system may also monitor and control thetemperature of the integrated circuit device 100, so that reactions takeplace and measurements are made at a known predetermined temperature.

The system may be configured to let a single fluid or reagent contactthe reference electrode 108 throughout an entire multi-step reactionduring operation. The valve 112 may be shut to prevent any wash solution110 from flowing into passage 109 as the reagents 114 are flowing.Although the flow of wash solution may be stopped, there may still beuninterrupted fluid and electrical communication between the referenceelectrode 108, passage 109, and the microwell array 107. The distancebetween the reference electrode 108 and the junction between passages109 and 111 may be selected so that little or no amount of the reagentsflowing in passage 109 and possibly diffusing into passage 111 reach thereference electrode 108. In an exemplary embodiment, the wash solution110 may be selected as being in continuous contact with the referenceelectrode 108, which may be especially useful for multi-step reactionsusing frequent wash steps.

FIG. 2 illustrates cross-sectional and expanded views of a portion ofthe integrated circuit device 100 and flow cell 101. The integratedcircuit device 100 includes the microwell array 107 of reaction regionsoperationally associated with sensor array 205. During operation, theflow chamber 105 of the flow cell 101 confines a reagent flow 208 ofdelivered reagents across open ends of the reaction regions in themicrowell array 107. The volume, shape, aspect ratio (such as basewidth-to-well depth ratio), and other dimensional characteristics of thereaction regions may be selected based on the nature of the reactiontaking place, as well as the reagents, byproducts, or labelingtechniques (if any) that are employed.

The chemical sensors of the sensor array 205 are responsive to (andgenerate output signals related to) chemical reactions within associatedreaction regions in the microwell array 107 to detect an analyte ofinterest. The chemical sensors of the sensor array 205 may for examplebe chemically sensitive field-effect transistors (chemFETs), such asion-sensitive field effect transistors (ISFETs). Examples of chemicalsensors and array configurations that may be used in embodiments aredescribed in U.S. Patent Application Publication No. 2010/0300559, No.2010/0197507, No. 2010/0301398, No. 2010/0300895, No. 2010/0137143, andNo. 2009/0026082, and U.S. Pat. No. 7,575,865, each which areincorporated by reference herein.

FIG. 3 illustrates a schematic diagram of a portion of the integratedcircuit device 100 including sensor array 205 having multiple chemicalsensors coupled to the same reaction region. In the illustratedembodiment, sixteen chemical sensors and four reaction regions areillustrated, representing a small section of the sensor array 205 andmicrowell array 107 that can include millions of chemical sensors andreaction regions.

The integrated circuit device 100 includes an access circuit foraccessing the chemical sensors of the sensor array 205. In theillustrated example, the access circuit includes a row select circuit310 coupled to the sensor array 205 via row lines 311-314. The accesscircuit also includes column output circuit 320 coupled to the sensorarray 205 via column lines 321-328.

The row select circuit 310 and the column output circuit 320 areresponsive to timing and control signals provided by the arraycontroller 124 in FIG. 1 to select the various chemical sensors andoperate the sensor array 205 as described below. The array controller124 also provides a reference bias voltage to the reference electrode(See, FIG. 1, reference numeral 108) to bias the reagents flowing acrossopen ends of the reaction regions 380, 382, 384, 386 of the microwellarray 107 during operation.

In the illustrated embodiment, groups of four chemical sensors areoperationally associated with each of the reaction regions 380, 382,384, 386. Alternatively, the number of chemical sensors operationallyassociated with a single reaction region may be different than four.More generally, two or more chemical sensors may be operationallyassociated with a single reaction region. In some embodiments, thenumber of chemical sensors operationally associated with a singlereaction region may be greater than four, such as sixteen or more.

The group containing chemical sensors 331.1-331.4 is representative ofthe groups of sensors of the sensor array 205. In the illustratedembodiment, each chemical sensor 331.1-331.4 includes achemically-sensitive field effect transistor 341.1-341.5 and a rowselect switch 351.1-351.4.

The chemically-sensitive field effect transistors 341.1-341.4 have acommon floating gate 370 in communication with the reaction region 380.That is, the common floating gate 370 is coupled to channels of each ofthe chemically-sensitive field effect transistors 341.1-341.5. Thechemically-sensitive field effect transistors 341.1-341.5 may eachinclude multiple patterned layers of conductive elements within layersof dielectric material.

The common floating gate 370 may for example include an uppermostconductive element (referred to herein as a sensor plate) that defines asurface (e.g. a bottom surface) of the reaction region 380. That is,there is no intervening deposited material layer between the uppermostelectrical conductor and the surface of the reaction region 380. In somealternative embodiments, the uppermost conductive element of the commonfloating gate 370 is separated from the reaction region 380 by adeposited sensing material (discussed in more detail below).

In operation, reactants, wash solutions, and other reagents may move inand out of the reaction region 380 by a diffusion mechanism. Thechemical sensors 331.1-331.4 are each responsive to (and generateindividual output signals related to) chemical reactions within thereaction region 380 to detect an analyte or reaction property ofinterest. Changes in the charge within the reaction region 380 causechanges in the voltage on the common floating gate 370, which in turnchanges the individual threshold voltages of each of thechemically-sensitive field effect transistors 341.1-341.4 of the sensors331.1-331.4.

In a read operation of a selected chemical sensor 331.1, the row selectcircuit 310 facilitates providing a bias voltage to row line 311sufficient to turn on row select transistor 351.1. Turning on the rowselect transistor 351.1 couples the drain terminal of thechemically-sensitive transistor 341.1 to the column line 321. The columnoutput circuit 320 facilitates providing a bias voltage to the columnline 321, and providing a bias current on the column line 321 that flowsthrough the chemically-sensitive transistor 341.1. This in turnestablishes a voltage at the source terminal of the chemically-sensitivetransistor 341.1, which is coupled to the column line 322. In doing so,the voltage on the column line 322 is based on the threshold voltage ofthe chemically-sensitive transistor 341.1, and thus based on the amountof charge within the reaction region 380. Alternatively, othertechniques may be used to read the selected chemical sensor 331.1.

The column output circuit 320 produces an individual output signal forthe chemically-sensitive transistor 341.1 based on the voltage on thecolumn line 322. The column output circuit 320 may include switches,sample and hold capacitors, current sources, buffers, and othercircuitry used to operate and read the chemical sensors, depending uponthe array configuration and read out technique. In some embodiments, thecolumn output circuit 320 may include circuits such as those describedin U.S. Patent Application Publication No. 2010/0300559, No.2010/0197507, No. 2010/0301398, No. 2010/0300895, No. 2010/0137143, andNo. 2009/0026082, and U.S. Pat. No. 7,575,865, which were incorporatedby reference above.

The individual output signals of the other chemical sensors 331.2-331.4coupled to the reaction region 380 can be read out in a similar fashion.In doing so, the column output circuit 320 produces individual outputsignals for each of the chemical sensors 331.1-331.4.

The individual output signals for each of the chemical sensors331.1-331.4 can then be combined or otherwise processed by the arraycontroller 124 (or other data processor) to calculate a resultant, lownoise output signal for the group of chemical sensors 331.1-331.4. Forexample, the resultant output signal may be an average of the individualoutput signals. In such a case, the SNR of the resultant output signalcan be increased by as much as the square root of the number ofindividual output signals. In addition, the resultant output signal cancompensate for differences among the values of the individual outputsignals, caused by variations in performance of the chemical sensors331.1-331.4 which could otherwise complicate the downstream signalprocessing.

FIG. 4 is a flow chart of an example process for calculating a resultantoutput signal for a group of chemical sensors coupled to a singlereaction region. Other embodiments may perform different or additionalsteps than the ones illustrated in FIG. 4. For convenience, FIG. 4 willbe described with reference to a system that performs a process. Thesystem can be for example, the system of FIG. 1.

At step 400, a chemical reaction is initiated within a reaction regioncoupled to a group of two or more chemical sensors. The group ofchemical sensors may for example include respective chemically-sensitivefield effect transistors having a common floating gate in communicationwith the reaction region, as described above with respect to FIG. 3. Thechemical reaction may be a sequencing reaction, as described above.

At step 410, individual output signals are obtained from the chemicalsensors in the group. The individual output signals may for example beobtained by selecting and reading out the individual chemical sensorsusing the techniques described above. In some embodiments, flowing ofreagent(s) causes chemical reactions within the reaction region thatrelease hydrogen ions, and the amplitude of the individual outputsignals from the chemical sensors is related to the amount of hydrogenions detected.

At step 420, a resultant output signal for the group is calculated basedon one or more of the individual output signals. The resultant outputsignal may for example be an average of the individual output signals.Alternatively, other techniques may be used to calculate the resultantoutput signal.

At step 430, a characteristic of the chemical reaction is determinedbased on the resultant output signal. For example, the characteristic ofthe chemical reaction may be determined based on the value of theresultant output signal using the techniques disclosed in Rearick etal., U.S. patent application Ser. No. 13/339,846, filed Dec. 29, 2011,based on U.S. Prov. Pat. Appl. No. 61/428,743, filed Dec. 30, 2010, and61/429,328, filed Jan. 3, 2011, and in Hubbell, U.S. patent applicationSer. No. 13/339,753, filed Dec. 29, 2011, based on U.S. Prov. Pat. Appl.No. 61/428,097, filed Dec. 29, 2010, each of which were incorporated byreference above.

FIG. 5 illustrates a cross-sectional view of portions of two groups ofchemical sensors and their corresponding reaction regions according to afirst embodiment. In FIG. 5, the chemically-sensitive field effecttransistors 341.1, 341.2 of the chemical sensors 331.1, 331.2 in thegroup of sensors 331.1-331.4 coupled to the reaction region 380 arevisible. The chemically-sensitive field effect transistors 341.3, 341.4of the other chemical sensors 331.3, 331.4 in the group lie behind thiscross-section. Similarly, the cross-section of FIG. 5 shows thechemically-sensitive field effect transistors of two chemical sensors inthe group of chemical sensors that is coupled to the adjacent reactionregion 382. In this illustration, the select switches of the chemicalsensors, access lines and other connections are omitted for simplicity.

The chemical sensor 331.1 is representative of the group of chemicalsensors 331.1-331.4. In the illustrated example, thechemically-sensitive field effect transistor 341.1 of the chemicalsensor 331.1 is a chemically-sensitive field effect transistor(chemFET), more specifically an ion-sensitive field effect transistor(ISFET) in this example.

The chemically-sensitive field effect transistor 341.1 includes commonfloating gate 370 having a conductive element 520 coupled to thereaction region 380. The conductive element 520 is the uppermostfloating gate conductor (also referred to herein as a sensor plate) inthe common floating gate 370. In the embodiment illustrated in FIG. 5,the common floating gate 370 includes multiple patterned layers ofconductive material within layers of dielectric material 519.

In FIG. 5, the conductive element 520 electrically connects individualmulti-layer floating gate structures that extend over the channelregions of each of the chemically-sensitive field effect transistors341.1-341.4 of the group of chemical sensors 331.1-331.4. In doing so,the common floating gate 370 is shared among the chemical sensors331.1-331.4.

The chemically-sensitive field effect transistor 341.1 includes a sourceregion 521 and a drain region 522 within a semiconductor substrate 354.The source region 521 and the drain region 522 comprise dopedsemiconductor material having a conductivity type different from theconductivity type of the substrate 554. For example, the source region521 and the drain region 522 may comprise doped P-type semiconductormaterial, and the substrate may comprise doped N-type semiconductormaterial.

Channel region 523 separates the source region 521 and the drain region522. The common floating gate 370 includes a conductive element 551separated from the channel region 523 by a gate dielectric 552. The gatedielectric 552 may be for example silicon dioxide. Alternatively, otherdielectrics may be used for the gate dielectric 552.

As shown in FIG. 5, the reaction region 380 is within an openingextending through dielectric material 510 to the upper surface of theconductive element 520. The dielectric material 510 may comprise one ormore layers of material, such as silicon dioxide or silicon nitride. Theopening in the dielectric material 510 may for example have a circularcross-section. Alternatively, the opening may be non-circular. Forexample, the cross-section may be square, rectangular, hexagonal, orirregularly shaped. The dimensions of the openings within the dielectricmaterial 510, and their pitch, can vary from embodiment to embodiment.

In the illustrated embodiment, the upper surface 530 of the conductiveelement 520 is the bottom surface of the reaction region 380. That is,there is no intervening deposited material layer between the uppersurface 530 of the conductive element 520 and the reaction region 380.As a result of this structure, the upper surface 530 of the conductiveelement 520 acts as the sensing surface for the group of chemicalsensors 331.1-331.4. The conductive element 520 may comprise one or moreof a variety of different materials to facilitate sensitivity toparticular ions (e.g. hydrogen ions).

During manufacturing and/or operation of the device, a thin oxide of theelectrically conductive material of the conductive element 520 may begrown on the upper surface 530 which acts as a sensing material (e.g. anion-sensitive sensing material) for the group of chemical sensors331.1-331.4. For example, in one embodiment the conductive element 520may be titanium nitride, and titanium oxide or titanium oxynitride maybe grown on the upper surface 530 during manufacturing and/or duringexposure to solutions during use. Whether an oxide is formed depends onthe conductive material, the manufacturing processes performed, and theconditions under which the device is operated.

In the illustrated example, the conductive element 520 is shown as asingle layer of material. More generally, the conductive element 520 maycomprise one or more layers of a variety of electrically conductivematerials, such as metals or ceramics, depending upon the embodiment.The conductive material can be for example a metallic material or alloythereof, or can be a ceramic material, or a combination thereof. Anexemplary metallic material includes one of aluminum, copper, nickel,titanium, silver, gold, platinum, hafnium, lanthanum, tantalum,tungsten, iridium, zirconium, palladium, or a combination thereof. Anexemplary ceramic material includes one of titanium nitride, titaniumaluminum nitride, titanium oxynitride, tantalum nitride, or acombination thereof.

In some alternative embodiments, an additional conformal sensingmaterial (not shown) is deposited on the sidewall of the opening in thedielectric material 510 and on the upper surface 530 of the sensor plate520. In such a case, an inner surface of the deposited sensing materialdefines the reaction region 380. The sensing material may comprise oneor more of a variety of different materials to facilitate sensitivity toparticular ions. For example, silicon nitride or silicon oxynitride, aswell as metal oxides such as silicon oxide, aluminum or tantalum oxides,generally provide sensitivity to hydrogen ions, whereas sensingmaterials comprising polyvinyl chloride containing valinomycin providesensitivity to potassium ions. Materials sensitive to other ions such assodium, silver, iron, bromine, iodine, calcium, and nitrate may also beused, depending upon the embodiment.

In operation, reactants, wash solutions, and other reagents may move inand out of the reaction region 580 by a diffusion mechanism 540. Each ofthe chemical sensors 331.1-331.4 are responsive to (and generates anoutput signal related to) the amount of charge 524 proximate to theconductive element 520. The presence of charge 524 in an analytesolution alters the surface potential at the interface between theanalyte solution and the conductive element 520, due to the protonationor deprotonation of surface charge groups. Changes in the charge 524cause changes in the voltage on the floating gate structure 518, whichin turn changes in the threshold voltages of the chemically-sensitivetransistors 341.1-341.4 of each of the chemical sensors 331.1-331.4. Therespective changes in threshold voltages can be measured by measuringthe current through the respective channel regions (e.g. channel region523 of sensor 331.1). As a result, each of the chemical sensors331.1-331.4 can be operated to provide individual current-based orvoltage-based output signals on an array line connected to itscorresponding source region or drain region.

In an embodiment, reactions carried out in the reaction region 380 canbe analytical reactions to identify or determine characteristics orproperties of an analyte of interest. Such reactions can directly orindirectly generate byproducts that affect the amount of charge 524adjacent to the conductive element 520. If such byproducts are producedin small amounts or rapidly decay or react with other constituents,multiple copies of the same analyte may be analyzed in the reactionregion 380 at the same time in order to increase the individual outputsignals generated by the group of chemical sensors 331.1-331.4. In anembodiment, multiple copies of an analyte may be attached to a solidphase support 512, either before or after deposition into the reactionregion 380. The solid phase support 512 may be microparticles,nanoparticles, beads, solid or porous gels, or the like. For simplicityand ease of explanation, solid phase support 512 is also referred hereinas a particle. For a nucleic acid analyte, multiple, connected copiesmay be made by rolling circle amplification (RCA), exponential RCA,Recombinase Polymerase Amplification (RPA), Polymerase Chain Reactionamplification (PCR), emulsion PCR amplification, or like techniques, toproduce an amplicon without the need of a solid support.

In various exemplary embodiments, the methods, systems, and computerreadable media described herein may advantageously be used to processand/or analyze data and signals obtained from electronic orcharged-based nucleic acid sequencing. In electronic or charged-basedsequencing (such as, pH-based sequencing), a nucleotide incorporationevent may be determined by detecting ions (e.g., hydrogen ions) that aregenerated as natural by-products of polymerase-catalyzed nucleotideextension reactions. This may be used to sequence a sample or templatenucleic acid, which may be a fragment of a nucleic acid sequence ofinterest, for example, and which may be directly or indirectly attachedas a clonal population to a solid support, such as a particle,microparticle, bead, etc. The sample or template nucleic acid may beoperably associated to a primer and polymerase and may be subjected torepeated cycles or “flows” of deoxynucleoside triphosphate (“dNTP”)addition (which may be referred to herein as “nucleotide flows” fromwhich nucleotide incorporations may result) and washing. The primer maybe annealed to the sample or template so that the primer's 3′ end can beextended by a polymerase whenever dNTPs complementary to the next basein the template are added. Then, based on the known sequence ofnucleotide flows and on measured output signals from the chemicalsensors indicative of ion concentration during each nucleotide flow, theidentity of the type, sequence and number of nucleotide(s) associatedwith a sample nucleic acid present in a reaction region coupled to agroup of chemical sensors can be determined.

FIGS. 6 to 10 illustrate stages in a manufacturing process for forming adevice including multiple chemical sensors coupled to the same reactionregion according to a first embodiment.

FIG. 6 illustrates a structure 600 formed in a first stage. Thestructure 600 includes partially completed floating gate structures forthe field effect transistors of the chemical sensors. For example, thestructure 600 includes partially completed floating gate structure 618for the chemically-sensitive field effect transistor 341.1 of thechemical sensor 331.1.

The structure 600 can be formed by depositing a layer of gate dielectricmaterial on the semiconductor substrate 554, and depositing a layer ofpolysilicon (or other electrically conductive material) on the layer ofgate dielectric material. The layer of polysilicon and the layer gatedielectric material can then be etched using an etch mask to form thegate dielectric elements (e.g. gate dielectric 552) and the lowermostconductive material element (e.g. conductive element 551) of thefloating gate structures. Following formation of an ion-implantationmask, ion implantation can then be performed to form the source anddrain regions (e.g. source region 521 and a drain region 522) of thechemical sensors.

A first layer of the dielectric material 519 can then be deposited overthe lowermost conductive material elements. Conductive plugs can then beformed within vias etched in the first layer of dielectric material 519to contact the lowermost conductive material elements of the floatinggate structures. A layer of conductive material can then be deposited onthe first layer of the dielectric material 519 and patterned to formsecond conductive material elements electrically connected to theconductive plugs. This process can then be repeated multiple times toform the partially completed floating gate structures shown in FIG. 6.Alternatively, other and/or additional techniques may be performed toform the structure.

Forming the structure 600 in FIG. 6 can also include forming additionalelements such as array lines (e.g. row lines, column lines, etc.) foraccessing the chemical sensors, additional doped regions in thesubstrate 554, and other circuitry (e.g. select switches, accesscircuitry, bias circuitry etc.) used to operate the chemical sensors,depending upon the device and array configuration in which the chemicalsensors are implemented.

Next, conductive material 700 is formed on the structure illustrated inFIG. 6 to contact the partially completed floating gate structures. Anetch mask including mask elements 720, 722 is then formed on theconductive material 700, resulting in the structure illustrated in FIG.7.

The conductive material 700 includes one or more layers of electricallyconductive material. For example, the conductive material 700 mayinclude a layer of titanium nitride formed on a layer of aluminum, or alayer of titanium nitride formed on a layer of copper. Alternatively,the number of layers may be different than two, and other and/oradditional conductive materials may be used. Examples of conductivematerials that can be used in some embodiments include tantalum,aluminum, lanthanum, titanium, zirconium, hafnium, tungsten, palladium,iridium, etc., and combinations thereof.

The locations of the mask elements 720, 722 define the locations of thesensor plates for the chemically-sensitive field effect transistors ofthe corresponding groups of chemical sensors. In the illustratedembodiment, the mask elements 720, 722 comprise photoresist materialwhich has been patterned using a lithographic process. Alternatively,other techniques and materials may be used.

Next, the conductive material 700 is etched using the mask elements 700,722 as a mask, resulting in the structure illustrated in FIG. 8. Theetching process forms conductive elements 520, 810 in electrical contactwith the partially completed floating gate structures of a correspondinggroup of sensors. The conductive element 520 electrically connects thepartially completed floating gate structures for a group of sensors331.1-331.4, to complete the common floating gate 370 for this group ofsensors. Similarly, the conductive material element 810 completes thecommon floating gate for the adjacent group of sensors.

Next, the mask elements 700, 722 are removed and dielectric material 510is formed, resulting in the structure illustrated in FIG. 9. Thedielectric material 510 may comprise one or more layers of depositeddielectric material, such as silicon dioxide or silicon nitride.

Next, the dielectric material 510 is etched to form openings definingreaction regions 380, 382 extending to upper surfaces of the conductivematerial elements 520, 810, resulting in the structure illustrated inFIG. 10.

FIG. 11 illustrates a cross-sectional view of portions of two groups ofchemical sensors and their corresponding reaction regions according to asecond embodiment. In contrast to the embodiment shown in FIG. 5, thecommon floating gate for each group of chemical sensors includes asensor plate that is smaller than the bottom surface of thecorresponding reaction region.

In FIG. 11, the floating gate structure 1118 for the group of chemicalsensors 331.1-331.4 includes conductive element 1120 coupled to thereaction region 380. The conductive element 1120 is coupled to theconductive element 520 by conductive plug 1130. The conductive element1120 is the uppermost floating gate conductor in the floating gatestructure 1118, and thus acts as the sensor plate for the group ofchemical sensors 331.1-331.4.

In FIG. 11, an upper surface 1122 of the conductive element 1120 is aportion of the bottom surface of the reaction region 380. That is, thereis no intervening deposited material layer between the upper surface1122 of the conductive element 1120 and the reaction region 380. As aresult of this structure, the upper surface 1122 of the conductiveelement 1120 acts as the sensing surface for the group of chemicalsensors 331.1-331.4. In the illustrated embodiment, the conductiveelement 1120 is within the dielectric material 1160, such that the uppersurface 1122 of the conductive element 1120 is co-planar with the uppersurface of the dielectric material 1160. Alternatively, the conductiveelement 1120 may be formed on the upper surface of dielectric material1160, and thus protrude slightly into the reaction region 380.

As shown in FIG. 11, the upper surface 1122 of the conductive element1120 has a width 1125 that is the less than the width of the bottomsurface of the reaction region 380. As described in more detail below,having a small conductive element 1120 as the sensor plate can enablethe signal-to-noise ratio (SNR) of the individual output signals of thechemical sensors 331.1-331.4 to be maximized.

The amplitude of the desired signal detected by the chemical sensors331.1-331.4 in response to the charge 524 in an analyte solution is asuperposition of the charge concentration along the interface betweenthe conductive element 1120 and the analyte solution. Because the charge524 is more highly concentrated at the bottom and middle of the reactionregion 380, the width 1125 of the conductive element 1120 is a tradeoffbetween the amplitude of the desired signal detected in response to thecharge 524, and the fluidic noise due to random fluctuation between theconductive element 1120 and the analyte solution. Increasing the width1125 of the conductive element 1120 increases the fluidic interface areafor the chemical sensors 331.1-331.4, which reduces fluidic noise.However, since the localized surface density of charge 524 decreaseswith distance from the middle of the reaction region 380, the conductiveelement 1120 detects a greater proportion of the signal from areashaving lower charge concentration, which can reduce the overallamplitude of the detected signal. In contrast, decreasing the width 1122of the conductive element 1120 reduces the sensing surface area and thusincreases the fluidic noise, but also increases the overall amplitude ofthe detected signal.

For a very small sensing surface area, Applicants have found that thefluidic noise changes as a function of the sensing surface areadifferently than the amplitude of the desired signal. Because the SNR ofan individual output signal is the ratio of these two quantities, thereis an optimal width 1125 at which the SNR of the individual outputsignals from the chemical sensors 331.1-331.2 is maximum.

The optimal width 1125 can vary from embodiment to embodiment dependingon the material characteristics of the conductive element 1120 and thedielectric materials 510, 1160, the volume, shape, aspect ratio (such asbase width-to-well depth ratio), and other dimensional characteristicsof the reaction regions, the nature of the reaction taking place, aswell as the reagents, byproducts, or labeling techniques (if any) thatare employed. The optimal width may for example be determinedempirically.

FIGS. 12 to 14 illustrate stages in a manufacturing process for forminga device including multiple chemical sensors coupled to the samereaction region according to a second embodiment.

FIG. 12 illustrates a first stage of forming conductive plugs 1210, 1220extending through dielectric material 1200 to contact the conductiveelements 520, 810 of the structure illustrated in FIG. 8. The structurein FIG. 12 can be formed by removing the mask elements 720, 722 in FIG.8 and forming dielectric material 1200 on the resulting structure. Viascan then be etched through the dielectric material 1200, and metaldeposited within the vias. A planarization process (e.g. chemicalmechanical polishing) can then be performed to remove the depositedmetal from the upper surface of the dielectric material 1200 and formthe plugs 1210, 1220. Alternatively, other techniques may be used.

Next, conductive material 1300 is formed on the structure illustrated inFIG. 12. An etch mask including mask elements 1320, 1322 is then formedon the conductive material 1300, resulting in the structure illustratedin FIG. 13.

The conductive material 1300 may comprise one or more layers ofconductive material, such as those described above with respect theconductive material 700 of FIG. 7. The locations of the mask elements1320, 1322 define the locations of the sensor plates of the field effecttransistors of the corresponding groups of chemical sensors. In theillustrated embodiment, the mask elements 1320, 1322 comprisephotoresist material which has been patterned using a lithographicprocess. Alternatively, other techniques and materials may be used.

Next, the conductive material 1300 is etched using the mask elements1320, 1322 as a mask to form the conductive elements 1120, 1400.Dielectric material 1160 is then formed between the conductive elements1120, 1400, resulting in the structure illustrated in FIG. 14.

Next, dielectric material 510 is formed on the structure illustrated inFIG. 14. The dielectric material 510 is then be etched to form openingsdefining reaction regions 380, 382 extending to upper surfaces of theconductive elements 1120, 1400, resulting in the structure illustratedin FIG. 11. The dielectric material 1160 may comprise material differentthan that of dielectric material 510. For example, the dielectricmaterial 510 may comprise material (e.g. silicon oxide) which can beselectively etched relative to the material (e.g. silicon nitride) ofthe dielectric material 1160 when subjected to a chosen etch process. Insuch a case, the dielectric material 1160 can act as an etch stop duringthe etching process used to form the reaction regions 380, 382. In doingso, the dielectric material 1160 can prevent etching past the conductiveelements 1120, 1400, and thus can define and maintain the desired shapeof the reaction regions 380, 382.

FIGS. 15 to 18 illustrate stages in a manufacturing process for forminga device including multiple chemical sensors coupled to the samereaction region according to a third embodiment.

FIG. 15 illustrates a first stage of forming dielectric material 1160 onthe structure illustrated in FIG. 12. An etch mask including maskelements 1510, 1520, 1530 is then formed on the dielectric material1160, resulting in the structure illustrated in FIG. 15. As described inmore detail below, openings between the mask elements 1510, 1520, 1530define the locations of the sensor plates of the field effecttransistors of the corresponding groups of chemical sensors.

Next, the dielectric material 1160 is etched using the mask elements1510, 1520, 1530 as an etch mask to form openings 1610, 1620 within thedielectric material 1160, resulting in the structure illustrated in FIG.16. As shown in FIG. 16, the openings extend to the upper surfaces ofthe conductive plugs 1210, 1220.

Next, the mask elements 1510, 1520, 1530 are removed and conductivematerial 1800 is deposited on the structure illustrated in FIG. 16,resulting in the structure illustrated in FIG. 17. The conductivematerial 1800 may comprise one or more layers of conductive material,such as those described above with respect the conductive material 700of FIG. 7.

Next, a planarization process (e.g. CMP) is performed to remove theconductive material 1800 from the upper surface of the dielectricmaterial 1160, resulting in the structure illustrated in FIG. 18. Theplanarization process leaves remaining conductive material within theopenings 1610, 1620 to form the conductive elements 1120, 1400.

Next, dielectric material 510 is formed on the structure illustrated inFIG. 18. The dielectric material 510 can then be etched to form openingsdefining reaction regions 380, 382 extending to upper surfaces of theconductive elements 1120, 1400, resulting in the structure illustratedin FIG. 11.

FIGS. 19 to 21 illustrate stages in a manufacturing process for forminga device including multiple chemical sensors coupled to the samereaction region according to a fourth embodiment.

FIG. 19 illustrates a first stage of forming conductive plugs 1210, 1220extending through dielectric material 1900 to contact the conductiveelements 520, 810 of the structure illustrated in FIG. 8. As describedin more detail below, the dielectric material 1900, comprising one ormore layers of dielectric material, acts an etch stop during thesubsequent formation of the reaction regions 380, 382. The structure inFIG. 19 can be formed by removing the mask elements 720, 722 illustratedin FIG. 8 and forming the dielectric material 1900 on the resultingstructure. The plugs 1210, 1220 can then be formed using the techniquesdescribed above with reference to FIG. 12. Alternatively, othertechniques may be used.

Next, conductive elements 1120, 1400 are formed on the upper surface ofthe dielectric material 1900, resulting in the structure illustrated inFIG. 20. The conductive elements 1120, 1400 may be formed by depositingconductive material, forming an etch mask including mask elementsdefining the locations of the conductive elements 1120, 1400, andetching the conductive material using the mask elements as an etch mask.

Next, dielectric material 510 is formed on the structure illustrated inFIG. 20. The dielectric material 510 is then be etched to form openingsdefining reaction regions 380, 382 extending to upper surfaces of theconductive elements 1120, 1400, resulting in the structure illustratedin FIG. 21. As shown in FIG. 21, in this embodiment the reaction regions380, 382 extend below the upper surfaces of the conductive elements1120, 1400 to expose their side surfaces.

The dielectric material 1900 may comprise material different than thatof dielectric material 510. For example, the dielectric material 510 maycomprise material (e.g. silicon oxide) which can be selectively etchedrelative to the material (e.g. silicon nitride) of the dielectricmaterial 1900 when subjected to a chosen etch process. In such a case,the dielectric material 1900 can act as an etch stop during the etchingprocess used to form the reaction regions 380, 382. In doing so, thedielectric material 1900 can prevent etching below the conductiveelements 1120, 1400, and thus can define and maintain the shape of thereaction regions 380, 382.

Various embodiments may be implemented using hardware elements, softwareelements, or a combination of both. Examples of hardware elements mayinclude processors, microprocessors, circuits, circuit elements (e.g.,transistors, resistors, capacitors, inductors, and so forth), integratedcircuits, application specific integrated circuits (ASIC), programmablelogic devices (PLD), digital signal processors (DSP), field programmablegate array (FPGA), logic gates, registers, semiconductor device, chips,microchips, chip sets, and so forth. Examples of software may includesoftware components, programs, applications, computer programs,application programs, system programs, machine programs, operatingsystem software, middleware, firmware, software modules, routines,subroutines, functions, methods, procedures, software interfaces,application program interfaces (API), instruction sets, computing code,computer code, code segments, computer code segments, words, values,symbols, or any combination thereof. Determining whether an embodimentis implemented using hardware elements and/or software elements may varyin accordance with any number of factors, such as desired computationalrate, power levels, heat tolerances, processing cycle budget, input datarates, output data rates, memory resources, data bus speeds and otherdesign or performance constraints.

Some embodiments may be implemented, for example, using acomputer-readable medium or article which may store an instruction or aset of instructions that, if executed by a machine, may cause themachine to perform a method and/or operations in accordance with theembodiments. Such a machine may include, for example, any suitableprocessing platform, computing platform, computing device, processingdevice, computing system, processing system, computer, processor, or thelike, and may be implemented using any suitable combination of hardwareand/or software. The computer-readable medium or article may include,for example, any suitable type of memory unit, memory device, memoryarticle, memory medium, storage device, storage article, storage mediumand/or storage unit, for example, memory, removable or non-removablemedia, erasable or non-erasable media, writeable or re-writeable media,digital or analog media, hard disk, floppy disk, read-only memorycompact disc (CD-ROM), recordable compact disc (CD-R), rewriteablecompact disc (CD-RW), optical disk, magnetic media, magneto-opticalmedia, removable memory cards or disks, various types of DigitalVersatile Disc (DVD), a tape, a cassette, or the like. The instructionsmay include any suitable type of code, such as source code, compiledcode, interpreted code, executable code, static code, dynamic code,encrypted code, and the like, implemented using any suitable high-level,low-level, object-oriented, visual, compiled and/or interpretedprogramming language.

While the present invention is disclosed by reference to the preferredembodiments and examples detailed above, it is to be understood thatthese examples are intended in an illustrative rather than in a limitingsense. It is contemplated that modifications and combinations willreadily occur to those skilled in the art, which modifications andcombinations will be within the spirit of the invention and the scope ofthe following claims.

What is claimed:
 1. A method of operating a device, the methodcomprising: initiating a chemical reaction within a reaction regioncoupled to a group of two or more chemical sensors having a commonfloating gate in communication with the reaction region, each chemicalsensor in the group including a chemically-sensitive field effecttransistor (chemFET) and a row select transistor; obtaining individualoutput signals for a first chemFET of a first chemical sensor and for asecond chemFET of a second chemical sensor in the group of two or morechemical sensors by coupling the first chemFET to a first column lineand coupling the second chemFET to a second column line in response tobiasing a first row select transistor of the first chemical sensor and asecond row select transistor of the second chemical sensor; the firstrow select transistor and the second row select transistor coupled to afirst row line of a plurality of row lines; and calculating a resultantoutput signal for the first chemical sensor and the second chemicalsensor based on individual output signals of the first-chemFET and thesecond chemFET.
 2. The method of claim 1, further comprising determininga characteristic of the chemical reaction based on the resultant outputsignal.
 3. The method of claim 1, wherein calculating the resultantoutput signal comprises averaging two or more of the individual outputsignals.
 4. The method of claim 1, wherein the chemically-sensitivefield effect transistors include respective floating gate structurescoupled to one another via a conductive element.
 5. The method of claim4, wherein the respective floating gate structures include a pluralityof conductors electrically coupled to one another and separated bydielectric layers, and the conductive element is an uppermost conductorin the plurality of conductors of the respective floating gatestructures.
 6. The method of claim 1, wherein a width of an uppermostconductive element of the common floating gate is less than a width of abottom surface of the reaction region.
 7. The method of claim 1, whereina bottom surface of the reaction region includes an upper surface of aconductive element of the common floating gate.
 8. The method of claim7, wherein the conductive element comprises an electrically conductivematerial, and the upper surface of the conductive element includes anoxide of the electrically conductive material.
 9. The method of claim 1,wherein respective sensing surfaces of the chemically-sensitive fieldeffect transistors are defined by one or more conductive elements of thecommon floating gate.
 10. The method of claim 1, wherein initiating thechemical reaction comprises initiating a sequencing reaction.
 11. Themethod of claim 10, wherein initiating the sequencing reactioncomprises: disposing a plurality of template nucleic acids in thereaction region; and introducing known nucleotides into the reactionregion.
 12. The method of claim 11, further comprising determining anumber of known nucleotides incorporated into one of the templatenucleic acids based on the resultant output signal.
 13. The method ofclaim 1, further comprising: obtaining individual output signals for athird chemical sensor and for a fourth chemFET of a fourth chemicalsensor in the group of two or more chemical sensors by coupling thethird chemFET to the first column line and coupling the fourth chemFETto the second column line in response to biasing a third row selecttransistor of the third chemical sensor and a fourth row selecttransistor of the fourth chemical sensor; the third row selecttransistor and the fourth row select transistor coupled to a second rowline of a plurality of row lines.
 14. The method of claim 13, furthercomprising: calculating a resultant output signal for the third chemicalsensor and the fourth chemical sensor based on individual output signalsof the third-chemFET and the fourth chemFET.
 15. The method of claim 11,wherein disposing a plurality of template nucleic acids comprisesdisposing a plurality of template nucleic acids attached to a solidsupport.
 16. The method of claim 1, wherein the the reaction region isin communication with the floating gate, then after initiating achemical reaction, the method of claim 1 further comprises: sensing abyproduct of a nucleotide extension reaction by a change of surfacepotential of a sensing surface deposited on a top conductive element ofthe floating gate.
 17. The method of claim 16, wherein sensing thebyproduct of a nucleotide extension reaction comprises sensing thechange of surface potential resulting from hydrogen ion release duringan extension reaction.